Method of assaying the antioxidant activity of pure compounds, extracts and biological fluids

ABSTRACT

A direct comparisons of the antioxidant activities of various pure compounds (eg., Trolox®(6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), extracts and biological fluids (eg. human plasma) can be effected by means of standardisable, objective method of determining and measuring the antioxidative and oxidative radical scavenging activities of a natural or synthetic substance, including the measurement of an indicator reaction product gas, ethylene, in the reaction headspace using Selective Ion Flow Tube Mass Spectrometry (SIFT-MS). This method assays ethylene liberated from α-keto-γ-methiolbutyric acid (KMBA) on reaction with peroxy radicals, other radicals or other reactive oxygen species that can oxidise KMBA to ethylene. From the production rate and concentration of the ethylene, the total antioxidant activity of an added analyte in question and its rate of reaction with oxidative free radicals may be determined.

TECHNICAL FIELD

The present invention relates to a novel method of ethylene analysis forassaying the antioxidant activity of pure compounds, extracts andbiological fluids using Selective Ion Flow Tube Mass Spectrometry(SIFT-MS). SIFT-MS technology allows the real time measurement of tracegases generally to a few parts per billion detection limit in complexmixtures such as breath or the headspaces above samples of urine orblood. To use such technology, a small amount of sample gas isintroduced into a stream of helium and H₃O, NO⁺ or O₂ ⁺ precursor ionsbefore electronic separation. Mass and absolute concentration analysisof each constituent is completed and displayed within seconds. Thefreedom from electron bombardment, magnetic separation and complexsample preparation such as used in gas chromatography and conventionalmass spectrometry by the use of the technology of the present inventionwill allow rapid, uncomplicated, inexpensive, accurate, multiple analyteresolution.

BACKGROUND ART

The formation of reactive oxygen species in aerobic organisms is anunavoidable consequence of the coupling of oxidative phosphorylation ofADP with the reduction of molecular oxygen by four electrons to water.Other sources of oxidative radicals include miccrosomal andphotosynthetic electron transport chains, active phagocytosis, and theactivity of a variety of enzymes that produce different reactive speciesas intermediates.

The in vivo generation of oxidative free radicals results in thechemical degradation of cellular organelles, membranes,deoxyribosenucleic acids (DNA), and other structural elements as well asthe disruption of biochemical pathways, transduction and translationevents as well as genetic replication and repair. These effects may betranslated into tissue, and organ damage and malfunction leading to awide variety of diseases and the generation of malignancies. Thesedamaging changes may be produced in various tissues by trauma,environmental hazards, metabolic defects, inflammation or infections aswell as the natural responses to cellular aging, or natural and acquiredimmunity to foods, commensal microorganisms, environmental agents orsurveillance against spontaneously occurring tumourogenesis. Natural orsynthetic dietary or parenterally administered agents capable ofreducing or eliminating oxidative free radicals in cells and tissues arecurrently thought to protect against actual or potential oxidativedamage in vivo.

The measurement of free radical generation and oxidation as well asoxidative radical scavenging by naturally occurring or extraneousmolecules is currently both complex and time consuming. This measurementis made possible by the application of SIFT-MS technology to provide arapid, continuous, sensitive means of measuring oxidative free radicaland scavenging activities without calibration, standards or complexsample preparation.

The application of the SIFT-MS technology enables measuring thebiochemical production of oxidative free radicals in vivo or in vitro tothe capacity of oxidative radical scavengers to impede, inhibit orcompete with the generation or activity of oxidative free radicals.Consequently analytical system utilising SIFT-MS technology can be usedto calibrate and standardise other in vitro measurement techniques,monitor and quantify oxidative chemical generation and reactivity,monitor and quantify antioxidant generation and reactivity and determinethe relative rates of the generation and the relative reactivities ofthose systems.

Cells have evolved oxidative defences that involve specially adaptedenzymes as well as membrane-associated and aqueous phase molecules. Theproduction of reactive oxygen species in vivo does not necessarily implycellular damage but oxidative stress is thought to occur when theproduction of those oxidative radicals exceeds the scavenging,protective capacity of the endogenous antioxidants.

In vitro assays of oxidative free radical activity based on the timerequired to obtain maximum oxygen consumption have been described.Wayner et al “Quantitative measurement of the total, peroxylradical-trapping antioxidant capability of human blood plasma bycontrolled peroxidation”. FEBS Lett. 1985;187;33-37), “phycoerythrinemission fluorescence”. Glazer A N. “Fluorescence-based assay forreactive oxygen species: A protective role for carnitine”. FASEB J.1988;2:2487-91) and peroxyl radical oxidation ofα--keto-γ-methiolbutyric acid (KMBA) to ethylene by gas chromatograhy(Weston G W, et al. A rapid gas chromatographic assay for determiningoxyradical scavenging capacity of antioxidants and biological fluids.Free Radical Biology & Medicine 1998;24:480-93)

The method of the present invention is based on the known partialinhibition of ethylene formation in the presence of antioxidants thatcompete with KMBA for oxyradicals. This has been measured previously inthe headspace of a reaction vessel by gas chromatography to derive theTotal Oxyradical Scavenging Capacity Assay (TOSCA).

OBJECT OF THE INVENTION

An object of the present invention is to measure the concentration ofethylene as an assay for antioxidant activity using SIFT-MS technology.

DISCLOSURE OF THE INVENTION

In one form the invention is a method of determining, measuring andcomparing the oxidative radical activity in a natural or syntheticsubstance including the measuring by SIFT-MS technology of the oxidativefree radical and scavenging activities in a gas sample taken from theheadspace of the substance to be measured, comprising measuring theconcentration of ethylene as an assay for antioxidant activity toprovide a measurement of the concentration of the analyte to therebyindicate the total activity of an antioxidant and the rate of reactionof the antioxidant with the substrate, the method comprising

-   -   producing, mass selecting and accelerating precursor ions into a        stream of inert carrier gas,    -   injecting a mixture of the gas sample and the analyte into the        carrier gas/ion stream,    -   allowing the ethylene in the reaction mixture head space to        react with the selected precursor ions,    -   detecting, amplifying and analysing the amount and rate of        ethylene produced in the reaction mixture headspace as a measure        of the rate and amount of introduced analyte antioxidant        activity.

Preferably the trace elements in the gas sample react with the precursorions in the helium stream.

Preferably the partial pressure of ethylene in the gas sample iscalculated as part of the measurement of the rate and amount ofintroduced analyte.

Preferably the gas sample is introduced into the carrier gas/ion streamat a calibrated rate via a heated capillary inlet.

Preferably the concentration of each gas species of volatile organiccompounds in the gas mixture is calculated from the number densities ofthe precursor and product ions.

Preferably the number densities are measured by a second mass filter inconjunction with a particle multiplier and a software interface.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of the rate of reaction of KMBA oxidation by peroxylradicals in the presence of increasing concentrations of trolox acid.

FIG. 2 is a graph of the rate of reaction of KMBA oxidation by peroxylradicals in the presence of increasing concentrations of ascorbic acid.

FIG. 3 is a graph of the rate of reaction of KMBA oxidation by peroxylradicals in the presence of increasing concentrations of uric acid.

FIG. 4 is a graph of the rate of reaction of KMBA oxidation by peroxylradicals in the presence of increasing concentrations of glutathione(GSH) acid.

FIG. 5 is a graph of the reaction rate of KMBA oxidation by peroxyradicals in the presence of increasing amounts of human plasma.

FIG. 6 is a graph of the regression of TOSC values of trolox acid atdifferent concentrations.

FIG. 7 is a graph of the regression of TOSC values of ascorbic acid atdifferent concentrations.

FIG. 8 is a graph of the regression of TOSC values of glutathione (GSH)acid at different concentrations.

FIG. 9 is a graph of the regression of TOSC values of uric acid atdifferent concentrations.

FIG. 10 is a graph of the regression of TOSC values of human plasma atdifferent concentrations.

DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

In a highly preferred form, peroxyl radicals were generated by thethermal homolysis of AAPH (2,2′-Azobis(2-amidinopropane)dihydrochloride)at 35° C. and at temperatures between 35° C.-39° C. An assay was carriedout using 0.2 mM of substrate KMBA and 20 mM of AAPH in 100 mM ofphosphate buffer at pH 7.4. The technique may be extended to otherradicals such as hydroxy, OH and alkoxy and other reactive oxygenspecies such as HOCl and ONOO— that can oxidise KMBA to ethylene.

The TOSC values were measured for different concentrations ofantioxidants, trolox, ascorbic acid, glutathione, uric acid and humanplasma and are obtained from growth curves of ethylene production withtime. These curves are shown in FIGS. 1 through 5 for the statedantioxidant at varying concentrations. The area under each of thesegrowth curves is found for each concentration of the specifiedantioxidant. The ratio of this area, ∫SA, to the area under the controlcurve, ∫CA, provides the measure of the TOSCA according toTOSCA=100-(∫SA/∫CA)×100

The linear TOSC relationships with concentration derived by this methodfor each of the antioxidants are shown in FIGS. 6 through 10.

Modifications of the SIFT-MS technique for analyzing trace components ofgas mixtures have been described by Milligan D B, Wilson P F, Mautner MN, Freeman C G, McEwan M J, Clough T J, Sherlock R R. Real-Time,High-Resolution Quantitative Measurement of Multiple Soil Gas Emissions:Selected Ion Flow Tube Mass Spectrometry. J. Environ. Qual. 2002 31:515-524. SIFT-MS measures trace gases in complex mixtures such as air,breath and the headspace above liquids, allowing the analysis of asingle exhalation of breath in real time, giving immediate resultswithout the need for pre-concentration of the volatile gas compounds orcalibration using standards.

SIFT-MS utilizes selective chemical ionization, using precursor ionsgenerated by electron impact, by microwave discharge or by glowdischarge. The precursor ions are mass selected using a quadrupole massfilter to inject mass selected precursor ions into a stream of heliumcarrier gas and allowed to reach thermal equilibrium. Positive ornegative precursor ions may be chosen. The precursor ion must beunreactive with the bulk gas within which the trace species is carried,but react rapidly with the trace species of interest. O₂ ⁺ ions are usedto measure ethylene in this assay. The reaction vessel headspace sampleis introduced into the carrier gas stream at a calibrated rate via aheated capillary inlet, alternatively a mass flow controller orcalibrated leak valve could be used. Following this, the tracecomponents within the sample gas mixture undergo reaction with theprecursor ions in the helium bath gas. The concentration of a (or each)trace species VOC in the gas mixture is then calculated from theobserved number densities of the precursor and product ions as measuredby a second mass filter (quadrupole or time-of-flight mass spectrometer)in conjunction with a particle multiplier and specialized softwareinterface, library and data processor/analyser. In order to calculatethe actual partial pressure of the trace species it is essential to knowthe rate of and products formed by the reaction of the precursor ionwith the trace neutral under the conditions within the flow tube.

This SIFT-MS analysis of ethylene generated by the peroxy radicalreactivity with KMBA is performed in real time, with no samplepreparation, no calibration and without standards. In one preferredembodiment of the invention the assay provides an in-vitro method forthe sensitive, rapid and continuous, real time, absolute concentrationdetermination and quantification of oxidative radical activity of purecompounds, extracts and biological fluids as well as the antioxidantactivity of pure compounds, extracts and biological fluids. The methodmay also be used for in-vivo measurement of antioxidant activity.

In the present invention the reaction between O₂ ⁺ and ethylene that ismeasured is as follows.O₂ ⁺+C₂H₄→C₂H₄ ⁺O₂ k=1.0×10⁻⁹ cm³ molecule⁻¹ s⁻¹

The precursor and product ions are scanned over predetermined ranges ofmass-to-charge ratio, m/z, for a given time. For this invention, thedownstream analytical mass filter was switched between the m/z value ofthe precursor ion (m/z 32) and the m/z value of C₂H₄ ⁺ (m/z 28) ethyleneto target the chosen oxyradical/KMBA end product trace gas species. Thepartial pressure of ethylene in the sample is then calculatedimmediately, on line, from the precursor and product ion count rates. Inthis way, rapid changes in ethylene concentrations are monitored bySIFT-MS in sequentially obtained headspaces during, or at the end of theoxidative reaction.

The invention provides a SIFT-MS method for the determination,measurement and comparison of oxidative radical activity activity invitro and in vivo in both natural and synthetic, substances using anyoxidant or antioxidant. The SIFT-MS method may be automated to determineand measure oxidative radical activity and antioxidant activity in anynatural and/or synthetic substances.

The following table demonstrates the linear correlation between thedifferent assays and sample concentration. TOSCA-SIFT TOSCA-GC ORACTrolox (μM) 2-15 2-20 0-3 Uric Acid (μM) 5-20 2-25 0-4 Ascorbic Acid(μM) 5-30 5-50 0-2 GSH (μM) 10-75  10-75  —In the above table:SIFT = selected ion flow tube-mass spectrometry TOSC measurementGC = gas chromatography TOSC measurement(Winston et al 1998)ORAC = oxygen radical absorbance capacity TOSC measurement(Cao G,Alessio HM, Cutler RG. Oxygen radical absorbance capacity assay forantioxidants. Free Radic. Biol Med. 1993; 14: 303-11)

The following table is a comparison of relative total oxyradicalscavenging capacity (TOSC) values (on a per unit concentration basis) ofdifferent antioxidants calculated from the TOSCA-SIFT-MS and othermethods; TOSCA, ORAC, TRAP-1 (phycoerythrin) (Ghiselli, A.; Serafini,M.; Maiani, G.; Azzini, E.; Ferno-Luzzi, A. A. Free Radical Biol. Med.18: 29-36,1995) and TRAP-2 (oxygen electrode). (Wayner, D. D. M.;Burton, G. W.; Ingold, K. U.; Barclay, L. R. C.; Locke, S. J. Biochem.Biophys. Acta., 924:408-419,1996.). TRAP stands for Total peroxyradical-trapping antioxidant activity. SIFT-MS TOSCA ORAC TRAP-1 TRAP-2Trolox 1 1 1 1 1 Ascorbic acid 0.36 0.46 0.52 0.75 0.85 GSH 0.22 0.19 —— 0.18 Uric acid 1.08 0.70 0.92 0.85 0.65

The following table is a comparison of relative total oxyradicalscavenging capacity (TOSC) values (on a per unit weight basis) ofdifferent antioxidants calculated from the TOSCA-SIFT-MS and the othermethods of TOSCA and ORAC. SIFT-MS TOSCA ORAC Trolox 1 1 1 Ascorbic acid0.36 0.69 0.67 GSH 0.19 0.16 — Uric acid 1.18 0.95 1.44By reason of the present invention, no actual sampling of the substanceto be measured is required since the analysis is taken from a headspaceof the sample. The method also allows automation of the process and iscapable of measuring absolute concentrations and is therefore suitablealso for serum and biological fluids.

Having described preferred embodiments of the invention it will beapparent to those skilled in the art that various changes and ablationscan be made to the embodiments and yet still come within the generalconcept of the invention. All such changes and alterations are intendedto be included in the scope of this specification.

1 A method of determining, measuring and comparing the oxidative radicalactivity in a natural or synthetic substance including the measuring bySIFT-MS technology of the oxidative free radical and scavengingactivities in a gas sample taken from the headspace of the substance tobe measured, comprising measuring the concentration of ethylene as anassay for antioxidant activity to provide a measurement of theconcentration of the analyte to thereby indicate the total activity ofan antioxidant and the rate of reaction of the antioxidant with thesubstrate, the method comprising producing, mass selecting andaccelerating precursor ions into a stream of inert carrier gas,injecting a mixture of the gas sample and the analyte into the carriergas/ion stream, allowing the ethylene in the reaction mixture head spaceto react with the selected precursor ions, detecting, amplifying andanalysing the amount and rate of ethylene produced in the reactionmixture headspace as a measure of the rate and amount of introducedanalyte antioxidant activity.
 2. The method as claimed in claim 1,wherein the trace elements in the gas sample react with the precursorions in the helium stream.
 3. The method as claimed in claim 1, whereinthe partial pressure of ethylene in the gas sample is calculated as partof the measurement of the rate and amount of introduced analyte.
 4. Themethod as claimed in claim 1, wherein the gas sample is introduced intothe carrier gas/ion stream at a calibrated rate via a heated capillaryinlet.
 5. The method as claimed in claim 1, wherein the concentration ofeach gas species of volatile organic compounds in the gas mixture iscalculated from the number densities of the precursor and product ions.6. The method of claim 6, wherein the number densities are measured by asecond mass filter in conjunction with a particle multiplier and asoftware interface.